FernandoAlbericio
`DepartmentofOrganic
`Chemistry,
`UniversityofBarcelona,
`08028-Barcelona,Spain
`
`OrthogonalProtecting
`GroupsforN a-Aminoand
`C-TerminalCarboxyl
`FunctionsinSolid-Phase
`PeptideSynthesis
`
`a-aminogroupofone
`Abstract:Forthecontrolledsynthesisofeventhesimplestdipeptide,theN
`oftheaminoacidsandtheC-terminalcarboxylgroupoftheothershouldbothbeblockedwith
`suitableprotectinggroups.Formationofthedesiredamidebondcannowoccuruponactivationof
`thefreecarboxylgroup.Aftercoupling,peptidesynthesiscanbecontinuedbyremovalofeitherof
`a-aminogroupofanother
`thetwoprotectinggroupsandcouplingwiththefreeC-terminusorN
`protectedaminoacid.Whenthreefunctionalaminoacidsarepresentinthesequence,thesidechain
`oftheseresiduesalsohastobeprotected.Itisimportantthatthereis ahighdegreeofcompatibility
`betweenthedifferenttypesofprotectinggroupssuchthatonetypemayberemovedselectivelyin
`thepresenceoftheothers.Attheendofthesynthesis,theprotectinggroupsmustberemovedtogive
`thedesiredpeptide.Thus,itisclearthattheprotectionschemeadoptedisoftheutmostimportance
`andmakesthedifferencebetweensuccessandfailurein agivensynthesis.SinceR.B.Merrifield
`introducedthesolid-phasestrategyforthesynthesisofpeptides,thisprerequisitehasbeenreadily
`accepted.Thisstrategyisusuallycarriedoutusingtwomainprotectionschemes:thetert-
`tert-butylmethods.However,forthe
`butoxycarbonyl/benzylandthe9-flourenylmethoxycarbonyl/
`solid-phasepreparationofcomplexorfragilepeptides,aswellasfortheconstructionoflibraries
`ofpeptidesorsmallmoleculesusing acombinatorialapproach, arangeofotherprotectinggroups
`a-aminoand
`isalsoneeded.ThisreviewsummarizesotherprotectinggroupsforboththeN
`C-terminalcarboxylfunctions.©2000JohnWiley &Sons,Inc.Biopoly55:123–139,2000
`
`Keywords:chemicallibraries;combinatorialchemistry;orthogonalstrategy;peptidesynthesis;
`protectinggroup
`
`INTRODUCTION
`
`Peptidesynthesisisbasedontheappropriatecombi-
`nationofprotectinggroupstogetherwithanefficient
`methodfortheactivationofthecarboxylgroupprior
`1 Inthesolid-
`toreactionwiththeaminocomponent.
`phasestrategydevelopedbyMerrifield,theC-termi-
`nalprotectinggroupisinfact apolymericcarrierand,
`consequently,thesynthesisiscarriedoutonaninsol-
`ublesupport. 2 Thesolid-phasemethodnowdomi-
`
`natessyntheticpeptidechemistryandthemajorityof
`peptidesaremadeusingthistechnique.
`SinceMerrifielddescribedthesolid-phaseap-
`proach,onlytwoprotectionschemeshavebeen
`widelyadopted.Thetert-butoxycarbonyl(Boc)/ben-
`zyl(Bzl)strategydependsongraduatedacidlability.
`Thus,whiletheBocgroupisremovedbytrifluoro-
`aceticacid(TFA),Bzl,andrelatedprotectinggroups
`areremovedattheendofthesyntheticstrategywith
`strongacidssuchasHFortrifluoromethanesulfonic.
`
`Biopolymers(PeptideScience),Vol.55,123–139(2000)
`©2000JohnWiley &Sons,Inc.
`
`2
`
`123
`
`MPI EXHIBIT 1006 PAGE 1
`
`MPI EXHIBIT 1006 PAGE 1
`
`

`

`124
`
`Albericio
`
`FIGURE 1 Structure of acid-labile protecting groups.
`
`The main drawback associated with this strategy is a
`lack of flexibility. Long exposure of the peptide chain
`to TFA in the removal the Boc group can also cause
`premature removal of the benzyl protecting group.
`Furthermore, conditions for the removal of Bzl groups
`will always remove the Boc group. Finally, some
`peptides containing fragile sequences will not survive
`the strong acid conditions used to remove the Bzl
`groups. The alternative 9-fluorenylmethoxycarbonyl
`(Fmoc)/tert-butyl (t-Bu) strategy3,4 is based on the
`orthogonal concept5 in the sense that the two protect-
`ing groups belong to independent classes and are
`removed by different mechanisms. The two groups
`can be removed therefore in any order in the presence
`of the group. Orthogonal protection schemes are
`milder because the selective deprotection is governed
`by alternative cleavage mechanisms rather than by
`reaction rates. The Fmoc group6 is removed by pip-
`eridine through a b-elimination reaction and tBu is
`removed by acidolysis with TFA. Although this strat-
`egy has several advantages with respect to the Boc/
`Bzl approach, the conditions are still, in some cases,
`too harsh and can be incompatible with certain se-
`quences. Furthermore,
`the preparation of complex
`molecules such as cyclic or branched systems could
`require the use of other protecting groups.7 In this
`report, other protecting groups for the amino function
`as well as for the carboxyl one are discussed.
`
`Na-AMINO PROTECTING GROUPS
`
`Acid-Labile Protecting Groups
`
`Groups that can be removed through a milder acid in
`comparison to the Boc group are 2-(4-biphenyl)iso-
`(Bpoc),8
`triphenylmethyl
`(trityl,
`propoxycarbonyl
`Trt),9 and a,a-dimethyl-3,5-dimethoxybenzyloxycar-
`bonyl (Ddz)10 (Figure 1).
`
`The Bpoc group can be removed by treatment with
`0.2– 0.5% TFA* in CH2Cl2 and has been used in
`combination with t-Bu side-chain protecting groups in
`the early stage of solid-phase peptide synthesis
`(SPPS).11,12
`The main disadvantage of these derivatives is that
`most are obtained as oils that undergo an autocatalytic
`decomposition to the amino acid, CO2, and the Bpoc
`olefin and its dimer over a half-life of weeks.13 Thus,
`they have to be isolated and stored as cyclohexyl
`ammonium (CHA) or dicyclohexyl
`ammonium
`(DCHA) salts. Liberation of the free carboxyl com-
`pounds is not a straightforward process due to the
`extremely high acid lability of the protecting group.
`Recently, a convenient method for the preparation of
`the O-pentafluorophenyl (O-Pfp) esters has been re-
`ported.13 Most of these compounds are obtained as
`crystalline solids. Furthermore, the Pfp ester is in-
`tended both to protect the highly acid-labile urethane
`from autocatalytic cleavage by the free acid during
`storage and to activate the derivative for acylation
`during peptide coupling reactions. The preparation of
`the Pfp esters of Bpoc-amino acids was performed by
`first reacting the starting amino acid derivative with
`either Bpoc-phenyl carbonate, Bpoc-azide, or Bpoc-
`p-methoxycarbonylphenyl carbonate to form the free
`Bpoc-amino acids, which were immediately esterified
`with dicyclohexylcarbodiimide (DCC).
`SPS of several model peptides has been performed
`using Pfp esters of Bpoc-amino acids (4 equiv) in the
`presence of 1-hydroxybenzotriazole (HOBt; 4 equiv),
`an additive to form in situ the OBt esters and therefore
`to increase the reactivity, and diisopropylethylamine
`(DIEA, 8 equiv) to neutralize Pfp-OH and HOBt, both
`of which are sufficiently acidic to present a threat to
`the stability of the Bpoc group,
`in CH2Cl2/N,N-
`
`* When oxymethyl-containing resins such as poly(ethylene gly-
`col)-based resins are used, higher contents of TFA for this and other
`acid-labile protecting groups could be necessary because some of
`the TFA will be employed for the protonation of the oxymethyl
`moieties.
`
`MPI EXHIBIT 1006 PAGE 2
`
`MPI EXHIBIT 1006 PAGE 2
`
`

`

`Orthogonal Protecting Groups
`
`125
`
`FIGURE 2 Scheme for the alkylation of amide bonds. Adapted from Ref. 22.
`
`dimethylformamide (DMF) (1:1). Removal of the
`protecting group was achieved with 0.5% TFA in
`CH2Cl2 (1 3 3 min 1 1 3 15 min). In this regard, the
`use of scavengers such as benzyl mercaptan and thio-
`phenol has been also reported.14
`The Trt group can be removed in solution with 1%
`15 and 0.1M HOBt in trifluoroethanol
`TFA in CH2Cl2
`(TFE).16 To extend the application of Trt-amino acids
`in solid-phase synthesis, as well as their compatibility
`with hyperacid-labile resins and acid-sensitive bi-
`omolecules, the use of mild acid conditions was in-
`vestigated.17 The results indicate that 0.1M 7-aza-1-
`hydroxybenzotriazole (HOAt)/0.12M Me3SiCl in TFE,
`0.25M Me3SiCl in TFE, 0.2% TFA/1% H2O in CH2Cl2,
`and 3% trichloroacetic acid (TCA) in CH2Cl2 all quan-
`titatively remove the Trt group and are compatible with
`the 3-(4-hydroxymethylphenoxy)propionic acid (AB)
`linker. Furthermore, solutions of 0.2% TFA/1% H2O
`in CH2Cl2, and 3% TCA in CH2Cl2 are compatible
`with the most labile Riniker handle and the synthesis
`of oligonucleotide–peptide conjugates, respectively.
`The synthesis of Trt-amino acids can be carried out
`starting either from the amino acid or the methyl
`ester.9,18 The former method involves in situ trimeth-
`ylsilyl ester formation to protect the carboxylic acid
`followed by reaction with Trt-Cl. The latter method
`requires reaction with Trt-Cl followed by hydrolysis
`of the methyl ester with LiOH/H2O/CH3OH at 25–
`40°C. In our laboratories the second strategy is pre-
`ferred since the overall yields are better (.75% as
`opposed to 40 – 60%) and column chromatography is
`not required to obtain pure derivatives.19
`The main drawback associated with Trt-amino ac-
`ids is that these compounds couple with other amino
`acid derivatives in lower yields when compared with
`carbamate-based protected amino acids.9 SPPS with
`Trt-amino acids have proceeded in good yields
`when N-[(dimethylamino)-1H-1,2,3-triazolo[4,5]b]-
`pyridino-1-ylmethylene]-N-methylmethanaminium
`hexafluorophosphate N-oxide (HATU) in the presence
`of DIEA was used as the coupling reagent. Thus,
`several model peptides have been synthesized using
`
`single couplings with HATU/DIEA (4 equiv:8 equiv)
`for 20 min and with 3% TCA in CH2Cl2 (2 3 3 min)
`for removal of the Trt group.19
`The Trt group could be used in combination with
`an Fmoc strategy in order to avoid diketopiperazine
`(DKP) formation in a similar manner to that described
`earlier for a Boc strategy.20 This procedure avoids the
`presence of the free amino function of the penultimate
`residue and incorporates the third residue with neu-
`tralization in situ. Thus, in those sequences that are
`prone to DKP formation, the following experimental
`protocol should be used: (i) incorporation of the pen-
`ultimate residue as its Trt derivative, (ii9) selective
`detritylation with TFA/H2O/CH2Cl2 (2:1:97) (5 3 1
`min) for AB-type resins or (ii0) TFA/H2O/CH2Cl2
`(0.2:1:99; 5 3 1 min) for Riniker-based resin, and (iii)
`incorporation of the third residue as its Fmoc deriva-
`tive under in situ neutralization/coupling conditions
`mediated by 7-azabenzotriazol-1-yl-N-oxy-tris(pyrro-
`lidino)phosphonium hexafluorophosphate (PyAOP)/
`DIEA (5 equiv each) in DMF for 1 h.21
`The Trt protecting group has also been used in a
`strategy developed to successively alkylate each
`amide bond following its formation.22 Thus, after the
`coupling of an Fmoc-amino acid, the Fmoc group is
`removed and the N-terminal amino function is repro-
`tected with Trt by reaction with Trt-Cl in the presence
`of DIEA. Treatment of the Trt-peptide resin with
`lithium t-butoxide in tetrahydrofuran (THF) leads to
`the formation of the amide anion. Following removal
`of excess base, the alkylating reagent in DMSO is
`reacted with the Trt-peptide resin. The alkylation re-
`action mixture is then removed and the base and
`alkylation treatments repeated to drive the alkylation
`to completion. Removal of the Trt group, followed by
`coupling of the next Fmoc-amino acid allows the
`formation of the next amide bond, which can again be
`alkylated by following the same protocol involving
`the concourse of Trt group (Figure 2).
`The Ddz-amino acids, which are the most commer-
`cially available, are more stable in the presence of
`acids than both the Bpoc and the Trt analogues. The
`
`MPI EXHIBIT 1006 PAGE 3
`
`MPI EXHIBIT 1006 PAGE 3
`
`

`

`126
`
`Albericio
`
`FIGURE 3 Structure of protecting groups that are base labile through a a b-elimination reaction.
`
`Ddz group can be removed with 1–5% TFA in
`CH2Cl2 and the protected amino acids are described
`as being fully compatible with t-Bu side-chain pro-
`tecting groups.23 Furthermore, Ddz is also removed
`by photolysis at wavelengths above 280 nm. This
`property also makes the Ddz group potentially very
`useful in SPS library screening procedures. Ddz de-
`rivatives have a very characteristic uv absorption pat-
`tern with maxima at 230, 276, and 282 nm. Therefore,
`the Ddz fission product allows a very precise deter-
`mination of the initial loading of the first amino acid
`on the resin as well as of the growing Ddz-peptide
`resin.
`Ddz-amino acids have also been used to avoid
`DKP formation when a Backbone Amide Linker
`(BAL) strategy is used.24 As mentioned above for
`AB- and Riniker-type resins, the incorporation of the
`penultimate residue is performed using the Ddz de-
`rivative. Removal of the protecting group is achieved
`with TFA–H2O–CH2Cl2 (3:1:96) for 6 min before
`incorporation of the third residue as its Fmoc deriva-
`tive under in situ neutralization/coupling conditions
`mediated by PyAOP. The advantage of the Ddz pro-
`tecting group over the Trt group is that the former
`couples more efficiently, which is an especially im-
`portant factor when the penultimate residue has to be
`incorporated to a second and/or hindered amine.24
`
`Base-Labile Protecting Groups
`
`Although the use of Fmoc-based SPPS has increased
`enormously in recent years to the point where it is
`now probably the method of choice for the chemical
`synthesis of peptides, there could also be a need to
`search for other, base-labile amino protecting groups.
`The main drawbacks associated with the use of Fmoc-
`amino acids are related to their hydrophobicity. For
`example, some derivatives possess low solubility in
`the solvents commonly used in SPPS, which can
`cause low coupling yields and/or problems associated
`with automation. Solubility issues are more critical
`during the manipulation of Fmoc-protected peptides
`in a convergent strategy.25,26
`
`During the last few years, several Na-amino pro-
`tecting groups have been reported to overcome some
`of the problems described above. Examples of these
`are
`2-[4-(methylsulphonyl)phenylsulphonyl]ethyloxy-
`carbonyl (Mpc),27 2,2-bis-(49-nitrophenyl)ethyloxycar-
`bonyl (Bnpeoc),28 and 2-(2,4-dinitrophenyl)ethyloxycar-
`bonyl (Dnpeoc)29 (See Figure 3).
`Particularly appealing is the 2-(4-nitrophenylsulfo-
`nyl)ethoxycarbonyl (Nsc) group,27,30,31 which can be
`considered from the point of chemical composition to
`be a combination of the Dnpeoc and the Mpc groups,
`since both nitro and sulfonyl groups are incorporated
`into the structure. These two functional groups should
`enhance the solubility of an amino acid and prevent
`hydrophobic interactions between the peptide chains,
`a situation that should subsequently reduce the degree
`of failure or truncated sequences during chain elon-
`gation.32,33 Nsc-amino acids are synthesized easily
`from the corresponding succinimidyl carbonate and
`are crystalline compounds, a physical property
`strongly preferred for automated SPPS. Furthermore,
`the mechanism for the removal of the Nsc group is
`similar to that for Fmoc deprotection and is based
`upon a base-catalyzed b-elimination reaction. There-
`fore, the chemistry and instrumentation described for
`the Fmoc strategy can be readily adapted to this
`protecting group.
`The removal of the Nsc group proceeds at a rate
`three to ten times slower than the removal of the Fmoc
`group, and is dependent on the nature of the base and
`solvent (Table I).34 The high stability of Nsc in DMF
`is of particular importance when this group is to be
`left on for long period of time. Thus, the Nsc group is
`more suitable for use in automatic synthesizers where
`amino acid derivatives are stored in solution for rel-
`atively long periods or when protected peptides are
`left to react in a convergent strategy. In this sense,
`when traces of piperidine remain, the removal of
`Fmoc is still much faster.34 Nsc is clearly a better
`protecting group as far as stability is concerned. Con-
`ditions recommended for the removal of Nsc are 20%
`piperidine in DMF, or preferably DMF– dioxane (1:
`1), for 15 min. The addition of the stronger base
`
`MPI EXHIBIT 1006 PAGE 4
`
`MPI EXHIBIT 1006 PAGE 4
`
`

`

`Orthogonal Protecting Groups
`
`127
`
`Table I Comparison Between Fmoc and Nsc Protected Peptidesa
`
`Cleavage rate (t1/2)
`20% Piperidine/DMF
`1% DBU/20% piperidine/DMF
`Decomposition in DMF solution
`1 week
`3 weeks
`Olefin-amine adduct formation
`Polymerization during removal
`uv monitoring range
`
`a Source: Ref. 34.
`
`Fmoc
`
`10–15 s
`—
`
`Nsc
`
`90–110 s
`12–15 s
`
`10%
`40%
`Fast and reversible
`Yes
`302 nm
`
`, 1%
`2%
`Very fast and irreversible
`No
`380 nm
`
`1,8-diazabicyxlo[5.4.0]undec-7-ene (DBU) can com-
`pensate for the slower rate of Na-deprotection.30 In
`this case, caution is required because the use of DBU,
`even at a concentration of 1% (v/v), can cause aspar-
`timide formation in sensitive sequences.35 Other ad-
`vantages that Nsc has over the Fmoc group are that
`the formation of the olefin-amine adduct is irrevers-
`ible and takes place faster than with Fmoc.34 The Nsc
`group absorbs at 380 nm, which allows selective
`online monitoring during SPPS.
`Coupling kinetic studies have indicated that while
`Fmoc-amino acids at times coupled slightly faster
`than Nsc derivatives, both were globally within the
`same range of reactivity.36
`Premature removal of the Na-Fmoc protecting
`group has been observed during the synthesis of
`polyproline-containing peptides.37 This phenomenon
`is presumably due to the secondary amine character of
`the a-amino function of the Pro. Electrospray mass
`spectroscopy analysis of the sequence H–PPPPPPA–
`NH2 prepared with Nsc–Pro–OH showed no evidence
`of Pro insertion (detected as an [M 1 97] peak),
`which would be indicative of premature deprotection
`of a Pro and the subsequent coupling of another
`protected Pro.36
`
`Table II Racemization Studiesa
`
`H–Gly–aa–L-Phe–NH2
`
`The loss of configuration at the C-terminal car-
`boxyl residue is perhaps the most important side re-
`action in peptide synthesis.38 This is often enhanced
`by the use of highly potent coupling reagents, which
`usually convert the carboxyl function to a derivative
`bearing a good leaving group. Such leaving groups
`tend to increase the acidity of the a-proton and favor
`oxazolone formation, both of which lead to racemiza-
`tion.39 This risk is more accentuated when residues
`such as His,40 Cys,41,42 and Ser43 are incorporated
`into a sequence. To compare the racemization poten-
`tial of Nsc- and Fmoc-amino acids, the model tripep-
`tides H–Gly–Xxx–Phe–NH2 (where Xxx 5 His, Cys,
`and Ser)42,43 were manually synthesized by each
`method in the solid phase.36 Preactivation of the car-
`boxyl group was avoided to minimize racemization of
`the susceptible residues. High performance liquid
`chromatography (HPLC) analysis (Table II) revealed
`that for the Nsc-amino acids, racemization was unde-
`tected for Ser and reduced by more than a half for Cys
`and His. This illustrates the value of Na-Nsc Cys and
`His residues since Fmoc-amino acids gave unaccept-
`able results. Presumably, the higher stability of the
`Nsc-derivatives is correlated with the reduced acidity
`of the b-proton of the protecting group, which trans-
`
`aa: His
`
`aa: Cys
`
`aa: Ser
`
`% L,L
`
`97.5
`2.1
`98.9
`
`% D,L
`
`2.5
`97.9
`1.1
`
`% L,L
`
`89.9
`4.9
`95.7
`
`% D,L
`
`10.1
`95.1
`4.3
`
`% L,L
`
`98.9
`1.0
`99.9
`
`% D,L
`
`1.1
`99.0
`0.1
`
`Fmoc–L-aa–OH
`Fmoc–D-aa–OH
`Nsc–L-aa–OH
`
`a Source: Ref. 36.
`
`MPI EXHIBIT 1006 PAGE 5
`
`MPI EXHIBIT 1006 PAGE 5
`
`

`

`128
`
`Albericio
`
`FIGURE 4 Structure of Dde and ivDde protecting groups and the indazole derivative formed
`during the deprotection step.
`
`lates to a lower acidity of the a-proton of the amino
`acid. However, dedicated protocols for the automatic
`synthesis of peptides with a preactivation step using
`either Nsc or Fmoc chemistry should be developed.
`The use of weaker bases such as collidine (pKa 7.43)
`instead of DIEA (pKa 10.1) is a potential strategy as
`illustrated by studies performed on model sys-
`tems.42– 45
`In conclusion, the Nsc group can be considered an
`alternative to Fmoc for Na-protection.34,36,46 Nsc-
`amino acids are more suitable for use in automatic
`synthesizers where amino acid derivatives are stored
`in solution over relatively long periods or when pro-
`tected peptides are left to react in a convergent strat-
`egy. In this sense, the presence of an Nsc moiety, due
`to its polarity, should also facilitate the purification of
`protected peptides. Furthermore, the use of Nsc de-
`rivatives of Cys and His reduces the racemization of
`these residues when compared with the corresponding
`Fmoc derivatives. The preparation of proline-rich
`peptides occurs without premature removal of the Nsc
`group.
`Although the 1-(4,4-dimethyl-2,6-dioxocyclohexy-
`lidine)ethyl (Dde)47 group can be used for the Na-pro-
`tection, it has been mainly used for the protection of the
`«-amino function of Lys/Orn.48,49 Deprotection, which
`results in the formation of 3,6,6-trimethyl-4-oxo-4,5,6,7-
`tetrahydro-1H-indazole, is carried out by hydrazine–
`DMF (2:98) for 5 min, and can be monitored by the
`absorption at 300 nm. Dde is almost completely stable to
`conditions used to remove the Fmoc group. However,
`this small loss of Dde can compromise the purity of
`large peptides. Furthermore, Dde has also been reported
`3
`N9 migration from side-
`to undergo intramolecular N
`chain or a-amino group to the v-function of Lys/Orn,50
`resulting in the scrambling of the group within the pep-
`tide chain. In order to avoid both side reactions, the
`1-(4,4-dimethyl-2,6-dioxocyclohexylidine)-3-methylbu-
`tyl (ivDde)51 group has been described. This is removed
`in the same conditions than Dde. (See Figure 4.)
`Recently, a new family of Na-amino protecting
`groups, whose removal is achieved by nucleophilic
`addition to a Michael acceptor, has been de-
`
`scribed.52–54 The key deprotection step in the case of
`amines protected by either the 2-tert-butylsulfonyl-2-
`propenoxycarbonyl (Bspoc),53 the 1,1-dioxobenzo-
`(Bsmoc),52 or
`[b]thiophene-2-ylmethyloxycarbonyl
`the 2-methylsulfonyl-3-phenyl-1-prop-2-enyloxycar-
`bonyl (Mspoc)54 groups involves the addition of a
`nucleophilic reagent to the a,b-unsaturated sulfone
`system with the consequent ejection of the carbamate
`ion. (See Figures 5 and 6) There are several major
`advantages of such a process over the classic b-elim-
`ination process involved in the removal of groups
`such as Fmoc or Nsc: (i) the deblocking event is
`simultaneously a scavenging event,
`thus avoiding
`back alkylation of the b-elimination by-product, e.g.,
`dibenzofulvene52; (ii) lower concentrations of second-
`ary amine (piperidine or morpholine) can be used,
`thus minimizing base-catalyzed side reactions such as
`aspartimide formation52,55; and (iii) the method can
`be applied to the technique of rapid continuous solu-
`tion synthesis.55 In turn, the Mspoc group has some
`advantages with respect to the other two approaches
`in question. It is less sensitive to premature deblock-
`ing than the Bspoc and can be assembled from readily
`available materials without recourse to low-valent
`sulfur intermediates, as required in the case of the
`Bsmoc.54
`Due to the different lability in the presence of
`nucleophiles and the difference in the removal mech-
`anism, fluorenylmethyl (Fm)- and 1,1-dioxobenzo
`[b]thiophene-2-ylmethyl
`(Bsm)-based
`protecting
`groups can be orthogonally removed by the appropri-
`ate choice of deblocking reagent.52,55 Thus, Bsmoc
`can be removed in the presence of an Fm ester by
`treatment with 2% tris(2-aminoethyl)amine (TAEA)
`
`FIGURE 5 Structure of protecting groups that are base
`labile through a Michael addition.
`
`MPI EXHIBIT 1006 PAGE 6
`
`MPI EXHIBIT 1006 PAGE 6
`
`

`

`FIGURE 6 Mechanism for the removal of protecting
`groups that are base labile through a Michael addition.
`Adapted from Ref. 22.
`
`and Fmoc can be removed in the presence of a Bsm ester
`by N-methyl-tert-butylamine, the steric hindrance of
`which avoids extensive Michael-like addition.
`Several model peptides have been synthesized in
`solid-phase using Bsmoc-amino acids.52 The removal
`of the Bsmoc group was accomplished with 2–5%
`piperidine in DMF. The use of 2% piperidine in DMF
`for 7 min leads to a reduction in the extent of aspar-
`timide formation, e.g., 4.8 vs 11.6% obtained when
`20% piperidine is used for the removal of Fmoc group
`in a sequence prone to give this side reaction. An
`additional advantage of Bsmoc over Fmoc assembly
`was noted in the case of pentapeptide H–Tyr–Aib–
`Aib–Phe–Leu–OH, where the difficult Aib–Aib cou-
`pling was favored by about 13%.
`
`Pd-Labile Protecting Groups
`
`In 1950 the allyloxycarbonyl (Alloc) group was in-
`troduced by Stevens and Watanabe for the protection
`of amine and alcohol functions.56 They reported that
`cleavage of the Alloc group was accomplished by
`catalytic hydrogenolysis using platinum or palladium
`catalysts, the use of metallic sodium in liquid ammo-
`nia, or the use of phosphonium iodide in glacial acetic
`acid (HOAc). Unfortunately, these methods for the re-
`moval of the Alloc group were not straightforward,
`which limited its use in organic synthesis. With the
`palladium-catalyzed allyl removal reaction developed
`by Tsuji57 and Trost,58 the potential of the allyl protect-
`ing group was recognized.59,60 The deprotection step
`involves a palladium-catalyzed transfer of the allyl
`unit to various nucleophiles/scavengers in the pres-
`ence of a proton source (Figure 7). Thus, allyl-based
`groups are orthogonal with both base-labile, such as
`Fm, and acid-labile, such as t-Bu, protecting groups.
`
`Orthogonal Protecting Groups
`
`129
`
`Allyl chemistry has mainly been used in solid-
`phase strategies either as a handle or as a side-chain
`protecting strategy. Thus, Kunz and co-workers61 first
`introduced the use of 4-bromocrotonic acid coupled to
`aminomethyl polystyrene resin as an allylic anchor.
`Since then, other handles have also been used for the
`preparation of a broad range of peptides.25,60,62 Hud-
`son and co-workers63 and Loffet and Zhang64 inde-
`pendently investigated the use of the allyl group as a
`side-chain protecting group. The three-dimensional
`orthogonal
`strategy (Fmoc/t-Bu/allyl) has been
`mainly used for the preparation of cyclic peptides.65,66
`The Na-Alloc-amino acids are synthesized easily
`from the corresponding chloroformates67 using the
`method developed by Bolin and co-workers for the
`corresponding Fmoc derivatives.68 It should be men-
`tioned that even for hindered amino acids such as Val
`the formation of the protected dipeptide has been
`observed.69 This method involves the in situ prepara-
`tion of the trimethylsilyl derivatives and subsequent
`reaction with the chloroformate. Although these de-
`rivatives are obtained as oils, they can be crystallized,
`purified, and stored as their dicyclohexylammonium
`salts. Alternatively,
`the pentafluorophenyl esters,
`which are crystalline, can be easily prepared using the
`procedure described by Green and Berman for the
`Fmoc derivatives.70 These active esters represent an
`excellent method of storage for Na-Alloc amino acids
`and as active species can be used directly for synthetic
`purposes.67 In this case, it is convenient to perform
`the coupling in the presence of an equivalent of HOBt
`or HOAt in order to accelerate the process.
`The main concern with the use of the Alloc group
`for Na-amino protection is the formation of al-
`lylamines. The consequences of this side reaction can
`be more important when the Alloc group is used in a
`repetitive way for temporary Na-amino protection in
`SPPS. Allylamine formation can take place during the
`Pd-catalyzed removal of allylcarbamates and may oc-
`cur in different ways.60
`First of all, the free amine may compete with the
`nucleophilic allyl-group scavenger in the trapping of
`the p-allyl palladium complex. Therefore, it is better
`to use deprotection systems that lead to the protonated
`
`FIGURE 7 Palladium-catalyzed deprotection of allyl carbamates and carboxylates.
`
`MPI EXHIBIT 1006 PAGE 7
`
`MPI EXHIBIT 1006 PAGE 7
`
`

`

`130
`
`Albericio
`
`FIGURE 8 Mechanism of allylamine formation when protonic reversible allyl-group scavengers
`are used. Adapted from Ref. 60.
`
`amine or other masked non-nucleophilic forms of the
`amine, or to use the allyl scavenger in large excess.
`With the protonic reversible allyl-group scavengers
`ReH, allylamine may also be formed through an
`equilibration process (Figure 8). Finally, Alloc-
`amines are known to undergo decarboxylative rear-
`rangement to allyl amines in the presence of Pd cat-
`alysts and in the absence of nucleophiles.71
`The choice of an appropriate allyl scavenger is
`clearly a key factor for the establishment of a conve-
`nient strategy for the use of Na-Alloc as a temporary
`protecting group in SPPS. Guibe´ and co-workers72
`described the first SPPS using this protecting group.
`In this work, the removal of the Alloc group was
`effected by the hydrostannolytic procedure, which
`utilizes tributyltin hydride as the allyl group scaven-
`ger. This procedure was selected on the grounds that
`the hydrostannolytic procedure is extremely fast, be-
`ing complete within a few minutes (thus minimizing
`the risk of allylamine formation), and very selective,
`since tributyltin hydride is nonbasic and is, at room
`temperature, inert toward almost any reducible func-
`tions that do not interact with soluble palladium cat-
`alysts. On using this method the peptide was obtained
`in a moderate, nonoptimized yield of 25%. Two draw-
`
`backs, however, are associated with the use of tribu-
`tyltin hydride. The first is that tributyltin hydride is
`not easy to handle and, in particular, shows a propen-
`sity, in the presence of various agents including pal-
`ladium catalysts, to decompose into hexabutyldistan-
`nane and dihydrogen.72 The second drawback is as-
`sociated with the toxicity of tin compounds and the
`difficulty often encountered in their complete removal
`from the desired end products (this last difficulty is
`minimized in the case of synthesis on solid supports).
`Other allyl group scavengers have been investigated
`with the aim of developing alternative tin-free meth-
`ods that are well suited for repetitive Alloc removal
`and compatible with the presence of Fmoc and t-Bu
`protecting groups.67 Table III shows the results of a
`study carried out in which the Alloc derivative of the
`N-methylbenzylamine was taken as a model and
`shows a higher propensity to form allylamine than
`Alloc derivatives of primary amines. The study in-
`cludes the pronucleophilic species (NDMBA) and
`thiosalicyclic acid (TSA) proposed by Kunz73 and
`Geneˆt,74 respectively, and pseudometallic hydrides,
`2), initially proposed by Zhu
`i.e., borohydrides (BH4
`and co-workers,75 phenylsilane (PhSiH3) proposed by
`Guibe´ and co-workers,76 and several as yet untested
`
`Table III Palladium-Catalyzed Deprotection [Pd(Ph3)4] of the
`Alloc Derivative of N-Methylbenzylamine in the Presence of
`Various Allyl Group Scavengersa
`
`Allyl Group Scavenger
`
`Yield (%)
`HON(Me)CH2Ph
`AllON(Me)CH2Ph
`
`2
`
`NDMBA
`TSA
`PhSiH3
`Bu4N1BH4
`z BH3
`NH3
`(Me)2NH z BH3
`z BH3
`t-BuNH2
`(Me)3N z BH3
`Py z BH3
`
`a Source: Ref. 67.
`
`100
`100
`95
`60
`99.6 to 100
`100
`96 to 97
`0
`0
`
`0
`0
`5
`40
`0 to 0.4
`0
`3 to 4
`100
`100
`
`MPI EXHIBIT 1006 PAGE 8
`
`MPI EXHIBIT 1006 PAGE 8
`
`

`

`Orthogonal Protecting Groups
`
`131
`
`FIGURE 9 Palladium-catalyzed deprotection of allyl carbamates in the presence of boranes.
`Adapted from Ref. 67.
`
`z BH3,
`including NH3
`z BH3, (Me)3NH z BH3, and
`
`amine-borane complexes
`(Me)2NH z BH3, t-BuNH2
`Py z BH3.
`The pronucleophilic scavengers NDMBA and
`TSA, and the amine borane complexes H3N z BH3 and
`Me2NH z BH3, were found to lead selectively to
`N-methylbenzylamine. Meanwhile, concurrent and in-
`creasing formation of N-allyl- and N-methylben-
`z BH3
`zylamine was observed in the series t-BuNH2
`2. As far as the experiments with Py z
`, BH4
`# PhSiH3
`BH3 and Me3N z BH3 are concerned, these reactions
`led exclusively to the N-allylated amine. Clearly the
`tertiary amine– borane complexes do not act as allyl
`group scavengers at all. From the above experiments
`and with regard to selectivity, the pronucleophilic
`species NDMBA and TSA, on the one hand, and the
`amine– borane complexes H3N z BH3 and Me2NH z
`BH3, on the other hand, appear to be the best-suited
`scavengers for the palladium-catalyzed removal of
`N-Alloc groups. With regard to the reactivity, how-
`ever, the amine– borane complexes that lead to com-
`plete deprotection in less than 10 min are far superior
`to the pronucleophilic species, for which reaction
`times of between 30 and 90 min are required for the
`reaction to reach completion. Therefore, only the two
`amine– borane complexes H3N z BH3 and Me2NH z
`BH3 can be recommended as deprotecting agents for
`use in SPPS strategies. It should be noted that, in a
`similar way to that observed with other pseudometal-
`lic hydrides such as tributyltin hydride72 or phenylsi-
`lane,76,77 the deprotection of N-Alloc derivatives with
`amine– borane complexes does not lead to the free
`amines but to the corresponding boron carbamates,
`which are readily hydrolyzed and decarboxylated
`upon exposure, for instance, to small amounts of
`water or silica. (See Figure 9.)
`The stability of the Fmoc group and the ease of
`reduction of the Trp residue were assessed against
`these amine– borane complexes. It was found that
`Ac–Trp–OMe and Fmoc–Trp(Boc)–OMe were left
`unchanged after exposure at 25°C for 48 h to H3N z
`BH3 and Me2NH z BH3 either in the absence or the
`presence of Pd(Ph3)4. On the other hand, even in the
`absence of catalyst, Boc–Trp(CHO)–OMe is slowly